Immunohistochemical detection and quantitation of P-glycoprotein in multiple drug-resistant human myeloma cells: Association with level of drug resistance and drug accumulation

Using several multiple drug-resistant human myeloma cell lines as standards, we developed an immunohistochemical staining technique and means of quantitating P-glycoprotein in individual myeloma cells. The level of staining intensity of P-glycoprotein in individual myeloma cells was quantitated by measuring the average optical density of each cell with a microscopic computerized cell analysis system. Using this system, we observed that the level of P-glycoprotein for individual cells within a cell population of known drug sensitivity was very homogeneous (coefficient of variation, ≤ 13%). Analysis of cell lines with gradually increasing levels of multidrug resistance (8226/S, 8226/Dox₆ and 8226/Dox₄₀) demonstrated a close association between the level of resistance to doxorubicin, defined by the mean lethal dose (D₀) and the amount of P-glycoprotein on individual cells determined by the optical density (r = 0.82, P <0.0005). Intracellular doxorubicin (DOX) accumulation in the individual cell lines was inversely related to the level of drug resistance expressed as D₀. P-glycoprotein was also detected in the marrow-derived myeloma cells of patients with drug refractory disease using immunohistochemical staining. The amount of P-glycoprotein in the cells of one patient was directly compared to the amount found in the simultaneously stained standard cell lines (8226/Dox₆ and 8226/Dox₄₀) by comparing the optical densities for individual cells. Using this immunohistochemical technique to detect and quantitate P-glycoprotein in patient myeloma cells and comparing it to standard multidrug resistant myeloma cell lines may be of value in determining the contribution of P-glycoprotein to clinical drug resistance in patients with multiple myeloma.